Cloning of human C-type natriuretic peptide gene and reconstruction of its eukaryotic expressive vector.

XIAO Le; et al.

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Cloning of human C-type natriuretic peptide gene and reconstruction of its eukaryotic expressive vector.

更新时间：2024-01-17

- Cloning of human C-type natriuretic peptide gene and reconstruction of its eukaryotic expressive vector.
- Journal of Chongqing Medical University Issue 4, Pages: 474-477(2006)
- 作者机构：
  
  1. 重庆医科大学附属第一医院血管外科
  2. 重庆医科大学附属第一医院血管外科,重庆,400016
- 作者简介：
- 基金信息：
- DOI：
  CLC： Q78
- Published：2006，
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[1]肖乐,赵渝,时德.人C型利钠肽基因的克隆及其真核表达载体的构建[J].重庆医科大学学报,2006(04):474-477.

XIAO Le, et al.. Cloning of human C-type natriuretic peptide gene and reconstruction of its eukaryotic expressive vector.. [J]. Journal of Chongqing Medical University (4):474-477(2006)
[1]肖乐,赵渝,时德.人C型利钠肽基因的克隆及其真核表达载体的构建[J].重庆医科大学学报,2006(04):474-477. DOI：

XIAO Le, et al.. Cloning of human C-type natriuretic peptide gene and reconstruction of its eukaryotic expressive vector.. [J]. Journal of Chongqing Medical University (4):474-477(2006) DOI：

摘要

目的:为开展C型利钠肽基因治疗

克隆人C型利钠肽基因并构建其真核表达载体。方法:通过RT-PCR从人胎盘组织中克隆人C型利钠肽基因全长编码区

将该DNA片段亚克隆至克隆载体pBS-T中

用SmaⅠ单酶切筛选出负向插入片段

用HindⅢ和BamHⅠ双酶切将目的DNA片段定向克隆至真核表达质粒pcDNA3.1（+）中

然后用双酶切、PCR鉴定和DNA序列测定三重鉴定方法对重组质粒进行鉴定。结果:酶切、PCR及测序鉴定证实

人C型利钠肽基因全长编码区被准确插入至pcDNA3.1（+）酶切位点HindⅢ和BamHⅠ之间

并且未发现有碱基突变或移位。结论:含人C型利钠肽基因真核表达质粒的成功构建并鉴定

为开展C型利钠肽基因治疗奠定了物质基础。

Abstract

Objective: To clone human C-type natriuretic peptide gene and reconstruct its eukaryotic expressive vector for future investigation of gene therapy.Methods:The full coding domain sequence of human C-type natriuretic peptide gene was cloned from human placenta with RT-PCR and was sub-cloned into pBS-T.After screening of negative pBS-T/CNP with restriction enzyme SmaⅠdegestion

the targeted DNA fragment

digested with HindⅢand BamHⅠ

was directional cloned into eukaryotic expressive plasmid pcDNA3.1(+).Then

the reconstructed plasmid was identified with enzyme digestion

PCR and sequencing.Results:It was demonstrated that the full coding domain sequence of human C-type natriuretic peptide gene was accurately cloned into digestion sites between HindⅢand BamHⅠ in pcDNA3.1(+) without mutation and transposition.Conclusion:The successive reconstruction and verifying of eukaryotic expressive plasmid containing human C-type natriuretic peptide gene established the foundation of gene therapy of C-type natriuretic peptide.

关键词

C型利钠肽真核表达质粒基因治疗

Keywords

C-type natriuretic peptideEukaryotic expressive vectorgene therapy

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